The major goals of this project are to elucidate the molecular sites and mechanisms of action of ethanol on the N-methyI-D-aspartate class of ion channels. NMDA receptors are a subtype of glutamate-activated ligand-gated ion channel and they are involved in excitatory synaptic transmission in the brain. They mediate several forms of synaptic plasticity that underlie learning and memory and inhibition of their function by pharmacological or genetic means disrupts these complex behaviors. Acutely, ethanol inhibits ion flux through NMDA receptors while chronic exposure of neurons to ethanol induces an up-regulation in the functional status of these receptors that is manifested by signs of CNS excitability during withdrawal. Identifying the factors that regulate the inhibition of NMDA receptors by ethanol is important as acute sensitivity to the intoxicating effects of ethanol appears to be an important predictor of future alcohol abuse. Previous work carried out during this project identified several factors that can regulate the magnitude of inhibition of NMDA receptors to ethanol. These included subunit composition and post-translational modifications including phosphorylation and cytoskeleton interactions. In addition, this project identified an amino acid in the NR1 subunit of the receptor (F639) that when mutated to alanine significantly reduced the inhibitory effects of ethanol on NMDA receptors containing any NR2 subunit. In this application, three specific hypotheses are proposed to further define the molecular mechanisms that regulate the ethanol sensitivity of NMDA receptors. Aim 1 will extend our mutagenesis studies to examine additional mutations at F639 and homologous NR2 sites as well as residues in other TM domains. Aim 2 will investigate the effects of C-terminal phosphorylation of NR1 and NR2 residues on ethanol inhibition of NMDA receptors. Aim 3 will express ethanol-insensitive receptors identified in Aims 1 and 2 in neurons in vitro and in vivo to address which effects of ethanol are mediated by inhibition of NMDA receptors.